Updated on 2024/04/23

写真a

 
KANAO Tadayoshi
 
Organization
Faculty of Environmental, Life, Natural Science and Technology Professor
Position
Professor
External link

Degree

  • Doctor of Engineering ( 2002.11   Kyoto University )

Professional Memberships

 

Papers

  • Tetrathionate hydrolase from the acidophilic microorganisms. Reviewed International journal

    Tadayoshi Kanao

    Frontiers in microbiology   15   1338669 - 1338669   2024

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    Tetrathionate hydrolase (TTH) is a unique enzyme found in acidophilic sulfur-oxidizing microorganisms, such as bacteria and archaea. This enzyme catalyzes the hydrolysis of tetrathionate to thiosulfate, elemental sulfur, and sulfate. It is also involved in dissimilatory sulfur oxidation metabolism, the S4-intermediate pathway. TTHs have been purified and characterized from acidophilic autotrophic sulfur-oxidizing microorganisms. All purified TTHs show an optimum pH in the acidic range, suggesting that they are localized in the periplasmic space or outer membrane. In particular, the gene encoding TTH from Acidithiobacillus ferrooxidans (Af-tth) was identified and recombinantly expressed in Escherichia coli cells. TTH activity could be recovered from the recombinant inclusion bodies by acid refolding treatment for crystallization. The mechanism of tetrathionate hydrolysis was then elucidated by X-ray crystal structure analysis. Af-tth is highly expressed in tetrathionate-grown cells but not in iron-grown cells. These unique structural properties, reaction mechanisms, gene expression, and regulatory mechanisms are discussed in this review.

    DOI: 10.3389/fmicb.2024.1338669

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  • Recombinant expression using the tetrathionate hydrolase promoter in Acidithiobacillus ferrooxidans Reviewed

    Tadayoshi Kanao, Tomoki Kunihisa, Shuji Ohgimoto, Megumi Ito, Chisa Murakami, Hisayuki Nakayama, Takashi Tamura, Kazuo Kamimura

    J. Biosci. Bioeng.   135 ( 3 )   176 - 181   2023.3

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    In the iron- and sulfur-oxidizing acidophilic chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, tetrathionate hydrolase gene (Af-tth) is highly expressed during tetrathionate growth. The expression levels of Af-tth were specifically determined by quantitative reverse transcription-polymerase chain reaction and the expression ratios of S0/Fe2+ and S4O62-/Fe2+ were found to be 68 ± 21 and 181 ± 5, respectively. The transcriptional start site was identified by primer extension. Promoter regions of Af-tth were cloned into the expression shuttle vector pMPJC and GFP gene was under the direction of the regions. Green fluorescence was observed by UV irradiation in recombinant A. ferrooxidans harboring the plasmid colonies grown on tetrathionate. Furthermore, His-tagged Af-Tth was synthesized in the recombinant cells grown on tetrathionate. Recombinant, His-tagged Af-Tth in an active form, was rapidly purified through metal-affinity column chromatography, although recombinant Af-Tth was synthesized in the inclusion bodies of Escherichia coli and acid-refolding treatment was necessary to recover the activity. The specific activity of purified Af-Tth from recombinant A. ferrooxidans (2.2 ± 0.37 U mg-1) was similar to that of acid-refolded Af-Tth from recombinant E. coli (2.5 ± 0.18 U mg-1). This method can be applied not only to heterologous expression but also to homologous expression of target genes for modification or specific mutation in A. ferrooxidans cells.

    DOI: 10.1016/j.jbiosc.2022.12.005

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  • Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans. Reviewed International journal

    Tadayoshi Kanao, Naruki Hase, Hisayuki Nakayama, Kyoya Yoshida, Kazumi Nishiura, Megumi Kosaka, Kazuo Kamimura, Yu Hirano, Taro Tamada

    Protein science : a publication of the Protein Society   30 ( 2 )   328 - 338   2021.2

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    Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed β-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.

    DOI: 10.1002/pro.3984

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  • Multiple mutations in RNA polymerase β-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory. Reviewed International journal

    Saori Ogawa, Hitomi Shimidzu, Koji Fukuda, Naoki Tsunekawa, Toshiyuki Hirano, Fumitoshi Sato, Kei Yura, Tomohisa Hasunuma, Kozo Ochi, Michio Yamamoto, Wataru Sakamoto, Kentaro Hashimoto, Hiroyuki Ogata, Tadayoshi Kanao, Michiko Nemoto, Kenji Inagaki, Takashi Tamura

    Bioscience, biotechnology, and biochemistry   2021.1

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    Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

    DOI: 10.1093/bbb/zbab011

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  • Identification of a gene encoding a novel thiosulfate:quinone oxidoreductase in marine Acidithiobacillus sp. strain SH Reviewed

    Tadayoshi Kanao, Sultana Sharmin, Mirai Tokuhisa, Maho Otsuki, Kazuo Kamimura

    Research in Microbiology   171 ( 7 )   281 - 286   2020.10

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  • Draft Genome Sequence of Acidithiobacillus sp. Strain SH, a Marine Acidophilic Sulfur-Oxidizing Bacterium. Reviewed International journal

    Kazuo Kamimura, Sultana Sharmin, Eriko Yoshino, Mirai Tokuhisa, Tadayoshi Kanao

    Genome announcements   6 ( 6 )   2018.2

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    We announce here the genome sequence of a marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus sp. strain SH. The bacterium has potential for use in bioleaching of sulfide ores from seawater and contains a noble gene for thiosulfate quinone oxidoreductase in addition to specific genes for the oxidation of reduced inorganic sulfur compounds.

    DOI: 10.1128/genomeA.01603-17

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  • Characterization of tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH. Reviewed International journal

    Tadayoshi Kanao, Moe Onishi, Yasuyuki Kajitani, Yuki Hashimoto, Tatsuya Toge, Hiroshi Kikukawa, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   82 ( 1 )   152 - 160   2018.1

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    Tetrathionate hydrolase (4THase), a key enzyme of the S4-intermediate (S4I) pathway, was partially purified from marine acidophilic bacterium, Acidithiobacillus thiooxidans strain SH, and the gene encoding this enzyme (SH-tth) was identified. SH-Tth is a homodimer with a molecular mass of 97 ± 3 kDa, and contains a subunit 52 kDa in size. Enzyme activity was stimulated in the presence of 1 M NaCl, and showed the maximum at pH 3.0. Although 4THases from A. thiooxidans and the closely related Acidithiobacillus caldus strain have been reported to be periplasmic enzymes, SH-Tth seems to be localized on the outer membrane of the cell, and acts as a peripheral protein. Furthermore, both 4THase activity and SH-Tth proteins were detected in sulfur-grown cells of strain SH. These results suggested that SH-Tth is involved in elemental sulfur-oxidation, which is distinct from sulfur-oxidation in other sulfur-oxidizing strains such as A. thiooxidans and A. caldus.

    DOI: 10.1080/09168451.2017.1415128

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  • Characterization of a novel thiosulfate dehydrogenase from a marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH. Reviewed International journal

    Sultana Sharmin, Eriko Yoshino, Tadayoshi Kanao, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   80 ( 2 )   273 - 8   2016

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    A marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH, was isolated to develop a bioleaching process for NaCl-containing sulfide minerals. Because the sulfur moiety of sulfide minerals is metabolized to sulfate via thiosulfate as an intermediate, we purified and characterized the thiosulfate dehydrogenase (TSD) from strain SH. The enzyme had an apparent molecular mass of 44 kDa and was purified 71-fold from the solubilized membrane fraction. Tetrathionate was the product of the TSD-oxidized thiosulfate and ferricyanide or ubiquinone was the electron acceptor. Maximum enzyme activity was observed at pH 4.0, 40 °C, and 200 mM NaCl. To our knowledge, this is the first report of NaCl-stimulated TSD activity. TSD was structurally different from the previously reported thiosulfate-oxidizing enzymes. In addition, TSD activity was strongly inhibited by 2-heptyl-4-hydroxy-quinoline N-oxide, suggesting that the TSD is a novel thiosulfate:quinone reductase.

    DOI: 10.1080/09168451.2015.1088377

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  • Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan. Reviewed International journal

    Yang Wang, Takashi Yasuda, Sultana Sharmin, Tadayoshi Kanao, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   78 ( 7 )   1274 - 82   2014

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    Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51 mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis. The analysis of the obtained sequences showed their similarity to clones recently detected in other moderately acidic mine drainages. Uncultured bacteria related to Ferrovum- and Gallionella-like clones were dominant in the microbial community. Analyses using specific primers for acidophilic iron- or sulfur-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum spp., Acidithiobacillus caldus, Acidithiobacillus thiooxidans, and Sulfobacillus spp. revealed the absence of these bacteria in the microbial community in ARD from the Yasumi-ishi tunnel. Clones affiliated with a member of the order Thermoplasmatales were detected as the dominant archaea in the ARD microbial population.

    DOI: 10.1080/09168451.2014.915735

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  • The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity. Reviewed International journal

    Tadayoshi Kanao, Hisayuki Nakayama, Mizuki Kato, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   78 ( 12 )   2030 - 5   2014

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    Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.

    DOI: 10.1080/09168451.2014.948374

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  • Crystallization and preliminary X-ray diffraction analysis of tetrathionate hydrolase from Acidithiobacillus ferrooxidans. Reviewed International journal

    Tadayoshi Kanao, Megumi Kosaka, Kyoya Yoshida, Hisayuki Nakayama, Taro Tamada, Ryota Kuroki, Hidenori Yamada, Jun Takada, Kazuo Kamimura

    Acta crystallographica. Section F, Structural biology and crystallization communications   69 ( Pt 6 )   692 - 4   2013.6

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    Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity. The recombinant protein was crystallized in 20 mM glycine buffer pH 10 containing 50 mM sodium chloride and 33%(v/v) PEG 1000 using the hanging-drop vapour-diffusion method. The crystal was a hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 Å resolution and belongs to space group P3(1) or P3(2), with unit-cell parameters a = b = 92.1, c = 232.6 Å.

    DOI: 10.1107/S1744309113013419

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  • Tetrathionate-forming thiosulfate dehydrogenase from the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans. Reviewed International journal

    Mei Kikumoto, Shohei Nogami, Tadayoshi Kanao, Jun Takada, Kazuo Kamimura

    Applied and environmental microbiology   79 ( 1 )   113 - 20   2013.1

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    Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg(-1)) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg(-1)) observed at pH 2.5 and 50°C.

    DOI: 10.1128/AEM.02251-12

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  • Redox Transformations of Iron at Extremely Low pH: Fundamental and Applied Aspects. Reviewed International journal

    D Barrie Johnson, Tadayoshi Kanao, Sabrina Hedrich

    Frontiers in microbiology   3   96 - 96   2012

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    Many different species of acidophilic prokaryotes, widely distributed within the domains Bacteria and Archaea, can catalyze the dissimilatory oxidation of ferrous iron or reduction of ferric iron, or can do both. Microbially mediated cycling of iron in extremely acidic environments (pH < 3) is strongly influenced by the enhanced chemical stability of ferrous iron and far greater solubility of ferric iron under such conditions. Cycling of iron has been demonstrated in vitro using both pure and mixed cultures of acidophiles, and there is considerable evidence that active cycling of iron occurs in acid mine drainage streams, pit lakes, and iron-rich acidic rivers, such as the Rio Tinto. Measurements of specific rates of iron oxidation and reduction by acidophilic microorganisms show that different species vary in their capacities for iron oxido-reduction, and that this is influenced by the electron donor provided and growth conditions used. These measurements, and comparison with corresponding data for oxidation of reduced sulfur compounds, also help explain why ferrous iron is usually used preferentially as an electron donor by acidophiles that can oxidize both iron and sulfur, even though the energy yield from oxidizing iron is much smaller than that available from sulfur oxidation. Iron-oxidizing acidophiles have been used in biomining (a technology that harness their abilities to accelerate the oxidative dissolution of sulfidic minerals and thereby facilitate the extraction of precious and base metals) for several decades. More recently they have also been used to simultaneously remediate iron-contaminated surface and ground waters and produce a useful mineral by-product (schwertmannite). Bioprocessing of oxidized mineral ores using acidophiles that catalyze the reductive dissolution of ferric iron minerals such as goethite has also recently been demonstrated, and new biomining technologies based on this approach are being developed.

    DOI: 10.3389/fmicb.2012.00096

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  • Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans. Reviewed International journal

    Mohammed Abul Manchur, Mei Kikumoto, Tadayoshi Kanao, Jun Takada, Kazuo Kamimura

    Extremophiles : life under extreme conditions   15 ( 3 )   403 - 10   2011.5

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    An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.

    DOI: 10.1007/s00792-011-0371-6

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  • Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding. Reviewed International journal

    Tadayoshi Kanao, Chie Matsumoto, Kumiko Shiraga, Kyoya Yoshida, Jun Takada, Kazuo Kamimura

    FEMS microbiology letters   309 ( 1 )   43 - 7   2010.8

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    Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. The recombinant protein (Af-Tth) was successfully activated after an in vitro refolding treatment. The specific activity of the refolded Af-Tth obtained was 21.0+/-9.4 U mg(-1) when the protein solubilized from inclusion bodies by 6 M guanidine hydrochloride solution was refolded in a buffer containing 10 mM beta-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30% v/v glycerol with the pH adjusted to 4.0 by sulfuric acid for 14 h at 4 degrees C. The in vitro refolding experiments revealed that Af-Tth required exposure to an acidic environment during protein folding for activation. This property reflects a physiological characteristic of the Af-Tth localized in the outer membrane of the acidophilic A. ferrooxidans. No cofactor such as pyrroloquinoline quinone (PQQ) was required during the refolding process in spite of the similarity in the primary structure of Af-Tth to the PQQ family of proteins.

    DOI: 10.1111/j.1574-6968.2010.02019.x

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  • Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene. Reviewed

    Kazuo Kamimura, Ai Okabayashi, Mei Kikumoto, Mohammed Abul Manchur, Satoshi Wakai, Tadayoshi Kanao

    Journal of bioscience and bioengineering   109 ( 3 )   244 - 8   2010.3

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    Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.

    DOI: 10.1016/j.jbiosc.2009.08.007

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  • Reconstitution of iron oxidase from sulfur-grown Acidithiobacillus ferrooxidans. Reviewed International journal

    Taher M Taha, Tadayoshi Kanao, Fumiaki Takeuchi, Tsuyoshi Sugio

    Applied and environmental microbiology   74 ( 21 )   6808 - 10   2008.11

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    The iron oxidation system from sulfur-grown Acidithiobacillus ferrooxidans ATCC 23270 cells was reconstituted in vitro. Purified rusticyanin, cytochrome c, and aa(3)-type cytochrome oxidase were essential for reconstitution. The iron-oxidizing activity of the reconstituted system was 3.3-fold higher than that of the cell extract from which these components were purified.

    DOI: 10.1128/AEM.00787-08

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  • Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1. Reviewed International journal

    Tsuyoshi Sugio, Mitsuko Fujii, Yumika Ninomiya, Tadayoshi Kanao, Atsunori Negishi, Fumiaki Takeuchi

    Bioscience, biotechnology, and biochemistry   72 ( 7 )   1756 - 63   2008.7

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    Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.

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  • Isolation and characterization of Acidithiobacillus ferrooxidans strain D3-2 active in copper bioleaching from a copper mine in Chile. Reviewed International journal

    Tsuyoshi Sugio, Masanori Wakabayashi, Tadayoshi Kanao, Fumiaki Takeuchi

    Bioscience, biotechnology, and biochemistry   72 ( 4 )   998 - 1004   2008.4

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    Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu(2+) solubilized from 1% chalcopyrite (CuFeS(2)) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg x l(-1) x 30 d(-1). The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 microl O(2) uptake x mg protein(-1) x min(-1). In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 mul O(2) uptake.mg protein(-1).min(-1). Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.

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  • Effects of increasing concentration of salts on the expression of omp40 gene encoding a major porin of Acidithiobacillus ferrooxidans Reviewed

    Mohammed A. Manchur, Tadayoshi Kanao, Tsuyoshi Sugio, Kazuo Kamimura

    Journal of Biological Sciences   8 ( 1 )   125 - 130   2008.1

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    Effects of increasing concentration of NaCl, KCl and Na2SO4 on the growth of Acidithiobacillus ferrooxidans cells and on the expression of omp40 gene, encoding one of the major outer membrane proteins of A. ferrooxidans, were examined to obtain information on the response of A. ferrooxidans NASF-1 cells to environmental changes and to improve the bioleaching system operated in salt-containing environments. Although NaCl and KCl inhibited growth of A. ferrooxidans NASF-1 cells at the concentration of 0.3 M, A. ferrooxidans NASF-1 could grow in medium supplemented with 0.5 M Na2SO4. An analysis of outer membrane proteins by a polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate revealed that the relative amount of Omp40 was increased when cells were grown in Fe2+-medium supplemented with NaCl. Northern blot hybridization analyses also revealed that the transcription of omp40 gene was stimulated in cells incubated in medium supplemented with NaCl and KCl, but not with Na2SO4. © 2008 Asian Network for Scientific Information.

    DOI: 10.3923/jbs.2008.125.130

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  • Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans. Reviewed International journal

    Satoshi Wakai, Mizuho Tsujita, Mei Kikumoto, Mohammed A Manchur, Tadayoshi Kanao, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   71 ( 11 )   2735 - 42   2007.11

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    Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.

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  • Increase in Fe2+-producing activity during growth of Acidithiobacillus ferrooxidans ATCC23270 on sulfur. Reviewed International journal

    Tsuyoshi Sugio, Taher M Taha, Tadayoshi Kanao, Fumiaki Takeuchi

    Bioscience, biotechnology, and biochemistry   71 ( 11 )   2663 - 9   2007.11

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    When Acidithiobacillus ferrooxidans ATCC23270 cells, grown for many generations on sulfur were grown in sulfur medium with and without Fe(3+), the bacterium markedly increased not only in iron oxidase activity but also in Fe(2+)-producing sulfide:ferric ion oxidoreductase (SFORase) activity during the early log phase, and retained part of these activities during the late log phase. The activity of SFORase, which catalyzes the production of Fe(2+) from Fe(3+) and sulfur, of sulfur-grown cells was approximately 10-20 fold higher than that of iron-grown cells. aa(3) type cytochrome c oxidase, an important component of iron oxidase in A. ferrooxidans, was partially purified from sulfur-grown cells. A. ferrooxidans ATCC23270 cells grown for many generations on sulfur had the ability to grow on iron as rapidly as that did iron-grown cells. These results suggest that both iron oxidase and Fe(2+)-producing SFORase have a role in the energy generation of A. ferrooxidans ATCC23270 from sulfur.

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  • Identification of a gene encoding a tetrathionate hydrolase in Acidithiobacillus ferrooxidans. Reviewed International journal

    Tadayoshi Kanao, Kazuo Kamimura, Tsuyoshi Sugio

    Journal of biotechnology   132 ( 1 )   16 - 22   2007.10

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    Tetrathionate is one of the most important intermediates in dissimilatory sulfur oxidation and can itself be utilized as a sole energy source by some sulfur-oxidizing microorganisms. Tetrathionate hydrolase (4THase) plays a significant role in tetrathionate oxidation and should catalyze the initial step in the oxidative dissimilation when sulfur-oxidizing bacteria are grown on tetrathionate. 4THase activity was detected in tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells but not in iron-grown cells. A 4THase having a dimeric structure of identical 50kDa polypeptides was purified from tetrathionate-grown cells. The 4THase showed the maximum activity at pH 3.0 and high stability under acidic conditions. An open reading frame (ORF) encoding the N-terminal amino acid sequence of the purified 4THase was identified by a BLAST search using the database for the A. ferrooxidans ATCC 23270 genome. Heterologous expression of the gene in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. Antisera against the recombinant protein clearly recognized the purified native 4THase, indicating that the ORF encoded the 4THase.

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  • Involvement of iron oxidation enzyme system in sulfur oxidation of Acidithiobacillus ferrooxidans ATCC 23270 Reviewed

    Taher M. Taha, Tadayoshi Kanao, Fumiaki Takeuchi, Tsuyoshi Sugio

    BIOHYDROMETALLURY: FROM THE SINGLE CELL TO THE ENVIRONMENT   20-21   443 - +   2007

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    Growth of A. ferrooxidans ATCC 23270 cells in sulfur medium with 0.005% ferric sulfate for 3, 4, 5, 6, 7 and 10 days gave the maximum growth yield of 45, 58, 76, 86, 90 and 95 mg protein per liter medium, respectively. Iron oxidase activities of 1-, 2- and 3- day-cultured cells on sulfur with 0.005% ferric sulfate (3.4, 3.5 and 0.8 mu mol Fe2+ oxidized/mg protein/min) were approximately 68, 70 and 16% of iron-grown ATCC 23270 cells (5.0 mu mol/mg protein/min). In contrast iron oxidase activities of 1-, 2- and 3-day cultured cells on sulfur without iron (4.9, 3.8 and 2.7 mu mol Fe2+ oxidized/mg protein/min) were approximately 98, 76 and 54% of the iron oxidase activity observed in iron-grown ATCC 23270 cell. SFORase activities of 3 day-cultured cell on sulfur with and without ferric sulfate (0.62 and 0.31 mu mol Fe2+ produced/mg protein/min) were approximately 20 and 10 fold higher than that of iron-grown cell (0.03 mu mol Fe2+ produced/mg protein/min). Both iron oxidase and SFORase activities increased at early-log phase and decreased at late-lag phase during growth of the strain on sulfur with or without Fe3+. The plasma membranes which had iron oxidase activity were prepared not only from iron-grown cells but also sulfur-grown cells. Iron oxidase activities of the plasma membranes prepared from sulfur- and iron-grown cells were 3.6 and 4.5 nmol Fe2+ oxidized per mg protein per min. These results suggest that iron oxidation enzyme system has a role in part in the energy generation of this bacterium from sulfur.

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  • Phototrophic growth of a Rubisco-deficient mesophilic purple nonsulfur bacterium harboring a Type III Rubisco from a hyperthermophilic archaeon. Reviewed International journal

    Shosuke Yoshida, Masayuki Inui, Hideaki Yukawa, Tadayoshi Kanao, Ken-ichi Tomizawa, Haruyuki Atomi, Tadayuki Imanaka

    Journal of biotechnology   124 ( 3 )   532 - 44   2006.7

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    The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc(Tk)). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc(Tk), here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO2 fixation. Via double-crossover homologous recombination, we first deleted three Rubisco genes present on the chromosome of the photosynthetic mesophile Rhodopseudomonas palustris No. 7. The mutant strain (delta3) could neither grow under photoautotrophic nor photoheterotrophic conditions. We introduced the rbc(Tk) gene into strain delta3 either on a plasmid, or by integrating the gene onto the chromosome. The two transformant strains harboring rbc(Tk) displayed growth under photoautotrophic and photoheterotrophic conditions, both dependent on CO2 fixation. Specific growth rates and Rubisco activity levels were compared under photoheterotrophic conditions among the two transformants and the wild-type strain. We observed that the levels of Rubisco activity in the respective cell-free extracts correlated well with the specific growth rates. Immunoprecipitation experiments revealed that Rubisco activity detected in the transformants was derived solely from Rbc(Tk). These results demonstrated that the Type III Rbc(Tk) from a hyperthermophile could support CO2 fixation in a mesophilic organism, and that the specific growth rate of the transformant can be used as a convenient parameter for selection of engineered proteins with improved Rubisco activity.

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  • Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans. Reviewed International journal

    Tsuyoshi Sugio, Tomohiro Hisazumi, Tadayoshi Kanao, Kazuo Kamimura, Fumiaki Takeuchi, Atsunori Negishi

    Bioscience, biotechnology, and biochemistry   70 ( 7 )   1584 - 91   2006.7

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    It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

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  • Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine. Reviewed

    Ai Okabayashi, Satoshi Wakai, Tadayoshi Kanao, Tsuyoshi Sugio, Kazuo Kamimura

    Journal of bioscience and bioengineering   100 ( 6 )   644 - 52   2005.12

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    Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis. A molecular approach based on the construction of a 16S rDNA clone library was used to investigate the microbial population of the ARD. Small-subunit rRNA genes were PCR amplified, subsequently cloned and screened for variation by a restriction fragment length polymorphism (RFLP) analysis. A total of 284 clones were grouped into 133 operational taxonomic units (OTUs) by the RFLP analysis. Among them, an OTU showing the same RFLP pattern as those of the isolates from the ARD was not detected. The phylogenetic analysis based on the 16S rDNA sequences from 10 major OTUs and their close relatives revealed that 4 OTUs containing 32.1% of the total clones were loosely affiliated with Verrucomicrobia, 2 OTUs containing 6.6% of the total clones were loosely affiliated with Chloribi, and other OTUs were affiliated with Actinobacteria, Nitrospirae, and beta-Proteobacteria.

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  • Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans. Reviewed International journal

    Atsunori Negishi, Tadashi Muraoka, Terunobu Maeda, Fumiaki Takeuchi, Tadayoshi Kanao, Kazuo Kamimura, Tsuyoshi Sugio

    Bioscience, biotechnology, and biochemistry   69 ( 11 )   2073 - 80   2005.11

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    Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.

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  • Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon. Reviewed International journal

    Satoshi Wakai, Asami Ohmori, Tadayoshi Kanao, Tsuyoshi Sugio, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   69 ( 10 )   1884 - 91   2005.10

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    ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The K(m) value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45 degrees C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide. The genes encoding the subunits for the F1F(O)-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the alpha, beta, gamma, delta, and epsilon subunits of the F1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.

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  • Existence of an iron-oxidizing bacterium Acidithiobacillus ferrooxidans resistant to organomercurial compounds. Reviewed

    Fumiaki Takeuchi, Atsunori Negishi, Sosaku Nakamura, Tadayoshi Kanao, Kazuo Kamimura, Tsuyoshi Sugio

    Journal of bioscience and bioengineering   99 ( 6 )   586 - 91   2005.6

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    Acidithiobacillus ferrooxidans MON-1 which is highly resistant to Hg2+ could grow in a ferrous sulfate medium (pH 2.5) with 0.1 microM p-chloromercuribenzoic acid (PCMB) with a lag time of 2 d. In contrast, A. ferrooxidans AP19-3 which is sensitive to Hg2+ did not grow in the medium. Nine strains of A. ferrooxidans, including seven strains of the American Type Culture Collection grew in the medium with a lag time ranging from 5 to 12 d. The resting cells of MON-1, which has NADPH-dependent mercuric reductase activity, could volatilize Hg0 when incubated in acidic water (pH 3.0) containing 0.1 microM PCMB. However, the resting cells of AP19-3, which has a similar level of NADPH-dependent mercuric reductase activity compared with MON-1, did not volatilize Hg0 from the reaction mixture with 0.1 microM PCMB. The activity level of the 11 strains of A. ferrooxidans to volatilize Hg0 from PCMB corresponded well with the level of growth inhibition by PCMB observed in the growth experiments. The resting cells of MON-1 volatilized Hg0 from phenylmercury acetate (PMA) and methylmercury chloride (MMC) as well as PCMB. The cytosol prepared from MON-1 could volatilize Hg0 from PCMB (0.015 nmol mg(-1) h(-1)), PMA (0.33 nmol mg(-1) h(-1)) and MMC (0.005 nmol mg(-1) h(-1)) in the presence of NADPH and beta-mercaptoethanol.

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  • Effects of inhibitors and NaCl on the oxidation of reduced inorganic sulfur compounds by a marine acidophilic, sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH. Reviewed International journal

    Kazuo Kamimura, Emi Higashino, Tadayoshi Kanao, Tsuyoshi Sugio

    Extremophiles : life under extreme conditions   9 ( 1 )   45 - 51   2005.2

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    The effect of NaCl and the pathways of the oxidation of reduced inorganic sulfur compounds were studied using resting cells and cell-free extracts of Acidithiobacillus thiooxidans strain SH. This isolate specifically requires NaCl for growth. The oxidation of sulfur and sulfite by resting cells was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. Carbonylcyanide m-chlorophenyl-hydrazone and monensin were also relatively strong inhibitors. Thiosulfate-oxidizing activity was not inhibited by these uncouplers. Valinomycin did not inhibit the oxidation of sulfur compounds. NaCl stimulated the sulfur- and sulfite-oxidizing activities in resting cells but not in cell-free extracts. The tetrathionate-oxidizing activity in resting cells was slightly stimulated by NaCl, whereas it did not influence the thiosulfate-oxidizing activity. Sulfide oxidation was biphasic, suggesting the formation of intermediate sulfur. The initial phase of sulfide oxidation was not affected by NaCl, whereas the subsequent oxidation of sulfur in the second phase was Na+-dependent. A model is proposed for the role of NaCl in the metabolism of reduced sulfur compounds in A. thiooxidans strain SH.

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  • Involvement of sulfide:quinone oxidoreductase in sulfur oxidation of an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. Reviewed International journal

    Satoshi Wakai, Mei Kikumoto, Tadayoshi Kanao, Kazuo Kamimura

    Bioscience, biotechnology, and biochemistry   68 ( 12 )   2519 - 28   2004.12

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    The effects of cyanide, azide, and 2-n-Heptyl-4-hydroxy-quinoline-N-oxide (HQNO) on the oxidation of ferrous ion or elemental sulfur with Acidithiobacillus ferrooxidans NASF-1 cells grown in iron- or sulfur-medium were examined. The iron oxidation of both iron- and sulfur-grown cells was strongly inhibited by cyanide and azide, but not by HQNO. Sulfur oxidation was relatively resistant to cyanide and azide, and inhibited by HQNO. Higher sulfide oxidation, ubiquinol dehydrogenase activity, and sulfide:quinone oxidoreductase (SQR) activity were observed in sulfur-grown cells more than in iron-grown cells. Sulfide oxidation in the presence of ubiquinone with the membrane fraction was inhibited by HQNO, but not by cyanide, azide, antimycin A, and myxothiazol. The transcription of three genes, encoding an aa(3)-type cytochrome c oxidase (coxB), a bd-type ubiquinol oxidase (cydA), and an sqr, were measured by real-time reverse transcription polymerase chain reaction. The transcriptional levels of coxB and cydA genes were similar in sulfur- and iron-grown cells, but that of sqr was 3-fold higher in sulfur-grown cells than in iron-grown cells. A model is proposed for the oxidation of reduced inorganic sulfur compounds in A. ferrooxidans NASF-1 cells.

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  • Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase. Reviewed International journal

    Tadayoshi Kanao, Toshiaki Fukui, Haruyuki Atomi, Tadayuki Imanaka

    European journal of biochemistry   269 ( 14 )   3409 - 16   2002.7

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    The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an alpha(4)beta(4) heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent K(m) value of 0.21 +/- 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (n(H)) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 +/- 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a K(i) value of 0.037 +/- 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl-ACL. His273 of the alpha subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.

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  • Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle. Reviewed International journal

    Tadayoshi Kanao, Mineko Kawamura, Toshiaki Fukui, Haruyuki Atomi, Tadayuki Imanaka

    European journal of biochemistry   269 ( 7 )   1926 - 31   2002.4

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    Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2-oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2-fixation ability of the enzyme. The gene encoding Cl-IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary structure and the size of the recombinant protein indicated that Cl-IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 +/- 13 microm) and NADP+ (27 +/- 10 microm) were much smaller than those for 2-oxoglutarate (1.1 +/- 0.5 mm) and CO2 (1.3 +/- 0.3 mm). No significant differences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerevisiae (Sc-IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc-IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of Cl-IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl-IDH than Sc-IDH as a CO2-fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (K(i) = 0.35 +/- 0.04 mm) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.

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  • ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products Reviewed

    T Kanao, T Fukui, H Atomi, T Imanaka

    EUROPEAN JOURNAL OF BIOCHEMISTRY   268 ( 6 )   1670 - 1678   2001.3

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    The reductive tricarboxylic acid cycle functions as a carbon dioxide fixation pathway in the green sulfur bacterium, Chlorobium limicola. ATP-citrate lyase, one of the key enzymes of this cycle, was partially purified from C. limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. A DNA fragment was amplified with primers designed from this sequence and an internal sequence highly conserved among eukaryotic enzymes. Using this fragment as a probe, we isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant similarity to the N- and C-terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP-citrate lyase activity. The recombinant enzyme was purified from the cell-free extract of E. coli harboring aclBA for further characterization. The molecular mass of the recombinant enzyme was determined to be approximately 532-557 kDa by gel-filtration. The enzyme catalyzed the cleavage of citrate in an ATP-, CoA- and Mg2+-dependent manner, where ATP and Mg2+ could be replaced by dATP and Mn2+, respectively. ADP and oxaloacetate inhibited the reaction. These properties suggested that ATP-citrate lyase from C. limicola controlled the cycle flux depending on intracellular energy conditions. This paper provides the first direct evidence that a bacterial ATP-citrate lyase is a heteromeric enzyme, distinct from mammalian enzymes.

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  • Isolation and identification of an iron-oxidizing bacterium which can grow on tetrathionate medium and the properties of a tetrathionate-decomposing enzyme isolated from the bacterium Reviewed

    T Sugio, T Kanao, H Furukawa, T Nagasawa, RC Blake

    JOURNAL OF FERMENTATION AND BIOENGINEERING   82 ( 3 )   233 - 238   1996

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC FERMENTATION BIOENGINEERING, JAPAN  

    Among 150 pure strains of iron-oxidizing bacteria obtained from natural environments, two strains, Funis 2-1 and OK1-50, had the ability to use potassium tetrathionate (K2S4O6) as a sole energy source for growth. Funis 2-1 was a gram-negative, rod-shaped, acidophilic iron- and sulfur-oxidizing chemolithotrophic bacterium and had the same cytochrome composition and mean G+C content of DNA as Thiobacillus ferrooxidans, indicating that the strain is T. ferrooxidans. A tetrathionate-decomposing enzyme that catalyzes the disproportionate metabolism of 4 mol of tetrathionate into 7 mol of thiosulfate and 2 mol of sulfate was located on the plasma membrane of K2S4O6-grown, but not Fe2+-grown Funis 2-1 cells. Washed intact cells and cell extracts prepared from Funis 2-1 cells grown on K2S4O6 medium supplemented,vith more than 11 mM FeSO4 did not show K2S4O6-decomposing enzyme activity. K2S4O6-decomposing enzyme was purified to homogeneity from K2S4O6-grown Funis 2-1 cells. The apparent molecular weight of this enzyme was estimated to be 50,000 by gel filtration, 50,000 by SDS-PAGE, and 49,600 using a time-of-flight mass spectrometer, indicating that the enzyme is monomeric. The enzyme was most active at pH 3.5 and 50 degrees C and the activity was enhanced approximately 18 fold by a concentration of 200 mM of sulfate ion. The Michaelis constant of this enzyme for K2S4O6 was 0.73 mM.

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  • A DNA REGION THAT COMPLEMENTS ON ESCHERICHIA-COLI CYSG MUTATION IN THIOBACILLUS-FERROOXIDANS Reviewed

    T SUGIO, H SUZUKI, T TANAKA, S MATSUGI, K TANAKA, T KANAO, T TANO

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   59 ( 4 )   728 - 730   1995.4

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    Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space, The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3, When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysI), and AT2427 (cysJ), it complemented only the E, coli cysG mutation, Since cysG codes for S-adenosyl-L-methionine : uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen In methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.

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  • CO2 Utilization Technologies with Autotrophic Microorganisms

    ( Role: Joint author)

    2023.12  ( ISBN:9784781317564

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Research Projects

  • 無機硫黄化合物の酵素化学の確立とその応用

    Grant number:21K05876  2021.04 - 2025.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    金尾 忠芳

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

    鉄硫黄酸化細菌の一種 Acidithiobacillus ferrooxidans を研究の対象とし、硫黄化合物を代謝する酵素であるテトラチオン酸ハイドロラーゼ(4THase)について詳細な研究を行った。この4THaseは、無機硫黄化合物の加水分解反応を触媒し一部の好酸性微生物にのみ確認されている極めてユニークな酵素である。当該研究では既に、本菌の4THase遺伝子(Af-tth)の同定、組換え型酵素を結晶化し立体構造を決定した。さらに本酵素のユニークな反応メカニズムを解明するために、基質を浸漬した結晶構造解析を行うことにより、活性中心と反応に関わるアミノ酸残基の解析を行った。そしてD325がテトラチオン酸を加水分解するという、新規な反応メカニズムを解明した。さらにこの結晶構造解析から、D325の近傍に3つのメチオニン残基(M172, M238, M279)から成るメチオニンクラスターを発見し、これが硫黄原子を捕捉している様子を観察した。本研究課題においては、このメチオニンクラスターが本酵素活性にどの様に関わるのかを解明することに取り組み、最終的に反応メカニズムの完全解明を目指して研究を推進していく。一方で、Af-tth遺伝子がテトラチオン酸を生育基質とした場合に強く発現することから、この発現を担うプロモーターについても解析を行った。その結果、Af-tthの転写開始点とプロモーター領域を同定し、これを利用した本菌の組換え遺伝子発現ベクターを構築した。接合伝達による大腸菌とのシャトルベクターで、緑色蛍光タンパク質(GFP)遺伝子を本プロモーターの支配下においたベクタープラスミドをA. ferrooxidansに導入し、テトラチオン酸を生育基質とした組換え菌株は、薬剤耐性を持ち緑色蛍光を示した。これによりGFPのA. ferrooxidansを宿主とした組換え発現に成功した。

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  • 無機硫黄化合物の酵素化学の確立とその応用

    Grant number:17K08169  2017.04 - 2021.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    金尾 忠芳

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    鉄硫黄酸化細菌の一種 Acidithiobacillus ferrooxidans を研究の対象とし、硫黄化合物を代謝する酵素であるテトラチオン酸ハイドロラーゼ(4THase)について詳細な研究を行った。この4THaseは、無機硫黄化合物の加水分解反応を触媒し一部の好酸性微生物にのみ確認されている極めてユニークな酵素である。当該研究では既に、A. ferrooxidansの4THase遺伝子(Af-tth)を世界で初めて同定し、これを大腸菌組換え発現で得た封入体から酸性refolding処理により活性型酵素を獲得する新規な手法を開発し、その結晶化にも成功した。
    本研究にて得られたAf-Tth結晶をSPring-8を利用したX-線照射実験で1.75オングストロームの解像度で結晶構造データを回収することができた。基質のテトラチオン酸を浸漬した結晶のX-線照射データの解析から本酵素の反応機構を推測した。Af-Tthは他の硫黄代謝関連酵素で見られるCys依存性の反応機構とは全く異なり、Asp325の基質へのプロトン供与による加水分解メカニズムであることを解明し、部位特異的変異酵素(D325N)の活性の消失からこれを証明した。
    また、さらなるX-線結晶構造解析の結果、上記のAsp325付近から伸長する硫黄のオリゴマーと推定される電子密度、およびこれを捕捉する様な構造を持つ3つのメチオニン残基の存在を発見した。この3つのメチオニン残基は、本酵素における極めて特徴的な構造であり、他の好酸性微生物に由来する4THaseにも保存されている。まずはこのMet残基が捕捉している原子が硫黄原子であることを証明するためにSe-Met置換したAf-Tthの結晶を作成し、硫黄の吸収端に近い長波長X-線による解析を行なった。この結果、3つのメチオニン残基が捕捉した原子は単一の硫黄原子であることが分かった。

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  • Analyses of functions in marine sulfur-oxidizing bacterium and their application to bioleaching

    Grant number:16K08112  2016.04 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Kamimura Kazuo, Kanao Tadayoshi

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    Halophilic acidophilic sulfur-oxidizing bacteria have gained increasing interest because of their importance in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH is a marine bacterium with sodium chloride-stimulated sulfur-, sulfite-, and thiosulfate-oxidizing activities. The clarification of RISCs oxidation mechanism is indispensable to develop the efficient bioleaching. A novel thiosulfate:quinone oxidoreductase (TQO) was purified and characterized from strain SH. The gene for TQO was determined in the draft genome sequence of strain SH. The draft genome of strain SH provided further insights into the genomic diversity of members of the genus Acidithiobacillus, enabled us to determine a gene encoding the novel TQO, and contributed to a better understanding of the mechanism for RISCs metabolism in sulfur-oxidizing prokaryotes.

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  • Studies on the enzymatic chemistry involved in the inorganic sulfur metabolism and its application.

    Grant number:26450482  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Kanao Tadayoshi, TAMADA Taro, KAMIMURA Kazuo

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    Grant amount:\5070000 ( Direct expense: \3900000 、 Indirect expense:\1170000 )

    This study has been performed to progress our knowledge about the enzymes involved in the dissimilatory inorganic sulfur metabolism in sulfur-oxidizing bacteria. We have isolated and identified the gene encoding tetrathionate hydrolase (Af-tth) which is the key enzyme in the metabolism in sulfur-oxidizing bacterium, Acidithiobacillus ferrooxidans. The recombinant Af-Tth was successfully obtained as an active form by new refolding method under acidic condition. The enzyme was crystalized to apply X-ray diffraction analysis. The 3D structure of Af-Tth has been determined by MAD/SAD methods with the Se-methionylated enzyme. The active center was also determined by the X-ray diffraction analysis of the crystal with tetrathionate. No cysteine residue was observed around the active center in the enzyme. It suggested that the enzyme has a unique cysteine independent reaction distinct from almost all other enzymes concerning dissimilatory sulfur-metabolism.

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  • Studies on the enzymatic chemistry involved in the inorganic sulfur metabolism and its application

    Grant number:22580375  2010 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    KANAO Tadayoshi, KAMIMURA Kazuo

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    Grant amount:\4550000 ( Direct expense: \3500000 、 Indirect expense:\1050000 )

    This study has been performed to progress our knowledge about the enzymes involved in the dissimilatory inorganic sulfur metabolism in the sulfur-oxidizing bacteria. We have isolated and identified the gene encodingtetrathionate hydrolase (Af-tth) which is the key enzyme in the metabolism in Acidithiobacillus ferrooxidans. Although Af-tthwas cloned and expressed in recombinant Escherichia coli, the recombinant proteins were synthesized in inclusion bodies as an inactive form. However, we have developed a new method to obtain the recombinant Af-Tth in an active form by refolding under acidic conditions. The enzyme was purified to homogeneity and crystallized. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 A resolution and belongs to space group P31or P32with unit-cell parameters a = b= 92.1, c = 232.6 A. Further analyses such as multiple/single anomalous dispersion methods are required to determine the Af-Tth structure.

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  • Analysis of enzymes and genes involved in sulfur oxidation in a marine sulfur-oxidizing bacterium

    Grant number:18380194  2006 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    KAMIMURA Kazuo, KANAO Tadayoshi

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    Grant amount:\9970000 ( Direct expense: \9100000 、 Indirect expense:\870000 )

    鉄や硫黄を酸化してエネルギーを得る細菌を使用して, 硫化鉱石から銅やウランなどの金属を溶出させる技術にバクテリアリーチングがある。これまでの細菌は, NaCl存在下では増殖ができないため, 海洋環境などのようにNaClを含む環境下でのバクテリアリーチングは不可能だった。本研究では, そのような環境でバクテリアリーチングを可能にするために分離した細菌の, 硫黄酸化に関与する酵素や遺伝子の構造を明らかにした

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  • 二酸化炭素固定と余剰硫黄の有効利用を目指した鉄硫黄酸化細菌のエネルギー代謝解析

    Grant number:17780061  2005 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)  Grant-in-Aid for Young Scientists (B)

    金尾 忠芳

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    Grant amount:\3500000 ( Direct expense: \3500000 )

    本研究は、鉄硫黄酸化細菌Acidithiobacillus ferrooxidans 23270株をモデル微生物として、硫黄化合物のエネルギー代謝を解明することにより、石油の脱硫余剰硫黄を利用した炭酸固定などへの応用を最終目標として行われた。これまでにA.ferrooxidansはテトラチオン酸を基質に良好に生育することを明らかにし、テトラチオン酸を代謝するテトラチオン酸ハイドロラーゼ(4THase)を精製した。
    18年度は、本酵素の詳細な性質検討に取り組んだ。本酵素の反応最適pHは3.0、氷上60分間pH1.0においても95%以上の残存活性を示し、酸に対し極めて安定であることを明らかにした。反応生成物のHPLC分析より、本酵素は1分子のテトラチオン酸を加水分解して、1分子のチオ硫酸、元素硫黄、硫酸を生成することが分かった。本酵素のN-末端アミノ酸配列より同定したORFは1500bp,499AAをコードしており、N-末端より32アミノ酸は、配列からSec型のシグナルペプチドと推定された。このORFの大腸菌での発現を試みたが、組み換えタンパク質は不溶化して活性は検出できなかった。しかし、この抗体を用いてWestern blot解析を行った結果、このORFが4THase遺伝子であることを、分子量とN-末端アミノ酸配列だけでなく免疫学的にも証明した。4THase遺伝子の同定は世界で最初の研究であり、同時にデータベース上に類似したorthologueが存在しない本菌独自の新規な遺伝子の機能を同定した最初の研究にもなった。二価鉄、元素硫黄、テトラチオン酸と異なるエネルギー源で生育した本菌のWestern blot解析は、本酵素が硫黄化合物で生育した場合においてのみ発現する、本菌の硫黄のエネルギー代謝に深く関与していることを強く示唆した。
    なお、本研究内容に関する論文は現在投稿中である。

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